Toll-like receptors (TLRs) generate an innate immune signaling response upon recognizing broadly conserved microbial extracellular structures. Viral RNA is recognized by TLR3, TLR7 and TLR8, and microbial DNA is recognized by TLR9. Until recently the prevailing paradigm was that TLR9 recognized unmethylated CpG DNA motifs, which are abundant in bacteria but relatively scarce in mammalian DNA. However, recent studies including our own preliminary data suggest that TLR9 binds natural DNA ligands independently of their sequence and methylation state. We propose a comprehensive analysis of the structural properties that allow TLR9 to recognize microbial DNA including sequence, length, duplex content, methylation state, backbone chemistry (phosphodiester versus phosphorothioate), curvature and higher order structure (such as junctions). We show in preliminary studies that DNA curvature-inducing proteins HMGB1 and histones H2A and H2B significantly enhance binding to the C-terminal cleavage fragment of TLR9, suggesting that TLR9 preferentially recognizes curved DNA backbones. To determine the extent to which DNA curvature alone is responsible for the binding enhancement, we propose to measure the TLR9 binding affinity of DNA minicircles containing 75 to 120 base pairs. Since nucleosomes can induce TLR-dependent auto immunogenic signaling, we propose an in vitro biophysical analysis of whole nucleosomes as TLR9 ligands. These in vitro studies will be validated in vivo by measuring TLR9-dependent signaling responses in cells stimulated with various DNA or protein-DNA ligands including minicircles, nucleosomes, junctions and methylated DNA ligands. The TLR7/8/9 ectodomains must be proteolytically cleaved in order to produce receptors that are capable of signaling. In the first study with cleaved TLR9, we show in our preliminary data that both the N- and C-terminal TLR9 ectodomain fragments participate in ligand binding and receptor dimerization. We therefore hypothesize that the two fragments remain associated after proteolytic cleavage in the endosome, and that cleavage may be necessary for TLR9 to undergo the ligand-induced conformational change that activates the receptor. To test this hypothesis, we will explore the physical and functional relationships between the two TLR9 ectodomain fragments, and elucidate the physical basis of proteolytic activation using biophysical approaches. The lack of structural information for TLR7/8/9 limits our understanding of nucleic acid recognition by these receptors. We propose to use electron cryomicroscopy and X-ray crystallography as complementary approaches to gain insight into the structural basis of TLR9-DNA recognition. By providing a molecular-level understanding of the recognition of microbial DNA by TLR9, this project will provide the necessary tools to create more potent vaccine adjuvants, and a new class of anti-inflammatory therapeutics with a wide range of applications including in particular systemic lupus erythematosus, asthma, septic shock syndrome and organ transplant rejection.